(1) Field of the Invention
This invention relates to a diagnosis of sickle cell anemia and, more particularly, to a direct analysis of human hemoglobin genes by the use of restriction endonuclease Dde I and molecular hybridization procedures to detect sickle cell anemia.
(2) Description of the Prior Art
Sickle cell anemia is a debilitating genetic disorder that affects two of every thousand Blacks born in the United States. The disease is also prevalent in the Mediterranean area, as well as India. It is characterized by general weakness and pains in muscles and joints and can be fatal, frequently at an early age; victims do not live much beyond the age of 30. The disorder is caused by a single nucleotide base mutation in the globin gene which converts the glutamic acid codon (GAG) at amino acid position 6 to one for valine (GTG). That chemical abnormality produces red blood cells which are distorted into an inflexible sickle shape that can clog capillaries, blocking the flow of blood to the tissues.
The current test for sickle cell anemia diagnosis in fetuses is performed on samples of fetal blood obtained by fetoscopy. Through this procedure, a small sample of blood is taken from the fetus through the umbilical cord and then analyzed for sickle cells. The mutant protein migrates differently on the gel than the normal protein. While generally accurate, this diagnostic method is dangerous as it can cause a spontaneous abortion of the fetus 10 to 20% of the time.
Recombinant DNA techniques coupled with blot hybridization analysis have proven to be valuable tools for studying the molecular basis for hemoglobinopathies. Various researchers have used blot hybridization to confirm that .delta..beta.-thalassemia and hereditary persistence of fetal hemoglobin are the results of gene deletions, whereas .alpha.-thalassemia and .beta.-thalassemia are due to both gene deletions and point mutations. One study has shown that at least one case of .delta.-thalassemia is probably due to a base mutation.
These studies have also been extended to the clinical setting as methods for prenatal diagnosis of various genetic hematological conditions, generally the thalassemias. In addition, Kan and Dozy have reported, in Proc. Natl. Acad. Sci. USA 75, 5631-5635 (1978), the finding of a polymorphism for a Hpa I restriction endonuclease site in American Blacks 3' to the .beta. globin gene, which was shown to have a 60% association with the sickle cell allele. From their studies, they estimated that blot hybridization using this polymorphism alone could be successfully used for prenatal diagnosis of a sickle cell anemia in 36% of couples at risk. Phillips, et al. have combined the Hpa I analysis with a second polymorphism found in the .gamma. globin genes. In so doing, they have reported an extension of blot hybridization for prenatal diagnosis of sickle cell anemia to over 80% of the couples at risk. Proc. Natl'l. Acad. Sci. USA 77, 2853-2856 (1980). However, their analysis, which is safer than fetoscopies, requires family studies in order to establish the association of the polymorphic sites with the sickle cell allele. The analysis of polymorphisms requires analyzing DNA samples from both the mother and father to get 36% reliability and from both parents and one child already born to get 80% reliability. This limited application is a major disadvantage of these procedures.
A direct analysis of the sickle cell anemia should be possible by use of a restriction enzyme whose recognition sequence is created or eliminated by the sickle cell mutation. This approach would not require family studies, and should be useful for all couples at risk. Dr. A. Nienhuis has proposed such a direct analysis with restriction endonuclease Mnl I. N. Engl. J. Med. 299, 195-196 (1978). However, efforts in various laboratories have failed to attain the sensitivity requisite for the resolution by the DBM filter paper of the small [60-80 bp (base pairs)] fragments generated by this enzyme.